Anti-angiogenesis fusion protein and uses thereof

ABSTRACT

The present invention provides a bi-functional fusion protein, (AT)a-Fc-(VT)b, simultaneously targeting vascular endothelial growth factor (VEGF) and angiopoietins (ANGs), wherein AT is an ANG binding motif; and VT is a VEGF binding motif, Fragment crystallizable region (Fc) is an N-terminal of Immunoglobulin G (IgG); each of a and b is an integer from 1 to 10. The bi-functional fusion proteins contain two or more domains of human proteins and are of all human sequences, and thus are expected to be non-immunogenic, and potentially can be used therapeutically in human targeting angiogenesis-associated diseases.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of and priority to U.S. Provisional Application Ser. No. 62/719,377, filed Aug. 17, 2018, which is incorporated herein by reference in its entirety.

SEQUENCE LISTING

The present invention relates to an anti-angiogenesis fusion protein, which is a bi-functional fusion protein. A Sequence Listing, submitted as an ASCII text file via EFS-Web, is hereby incorporated by reference in accordance with 35 U.S.C. § 1.52(e). The name of the ASCII text file for the Sequence Listing is Aug. 19, 2019_SEQLIST_5992-0235PUS2.txt, the date of creation of the ASCII text file is Aug. 19, 2019, and the size of the ASCII text file is 35659 bytes.

FIELD OF THE INVENTION

The present invention relates to an anti-angiogenesis fusion protein, which is a bi- functional fusion protein.

BACKGROUND OF THE INVENTION

Angiogenesis, the formation of blood vessels by sprouting from pre-existing ones, is essential for the growth of solid tumors and for tumor metastasis. The generation of new capillaries involves a multistep process, which includes the dissolution of the membrane of the originating vessel, the endothelial cell migration and proliferation, and formation of a new vascular tube. Suppression of any one of these steps would inhibit the formation of new vessels and therefore affect tumor growth and generation of metastases. Indeed, it has been estimated that the elimination of a single endothelial cell could inhibit the growth of tumor cells. It has been reported that anti-angiogenic therapies may provide a promising mechanism for cancer treatment.

Vascular endothelial growth factor (VEGF) plays an important role in the process of angiogenesis. New blood vessel formation in diseases such as age-related macular degeneration (AMID) or tumor growth is mainly caused by the overexpression of this key growth factor [Connolly et al. (1989) J Clinic Invest 84(5):1470-8; Ferrara et al. (1989) Biochem Biophys Res Commun 161(2):851-8; Ferrara et al. (1989) Nat Med 4(3):336-40]. The VEGF family comprises several members such as VEGF-A, VEGF-B, VEGF-C, VEGF-D, and placenta growth factor (PGF). VEGF-A is the prototypic member of the VEGF family and also the most studied member. VEGF-A has been shown to stimulate endothelial cell mitogenesis, promote cell survival and proliferation, induce cell migration, and increase microvascular permeability. Targeting angiogenesis process through blocking VEGF activities with antibodies, VEGF traps, or VEGF receptor (VEGFR) kinase inhibitor have been demonstrated to be effective preclinically and clinically to treat cancers, and AMD disease as well [Major et al. (1997) J Pharmacol Exp. Ther 283(1):402-10; Willett et al. (2004) Nat Med 10(2):145-7; Papadopoulos et al. (2012) Angiogenesis 15(2): 171-85; Aiello et al.(1995) Proc. Natl. Acad. Sci. USA 92(23):10457-61]. Among VEGF blockers, Eylea, for example, is an engineered chimeric Fc fusion protein containing the VEGFR1 extracellular Ig domain 2 (ECD, D2), VEGFR2 extracellular Ig domain 3 (ECD, D3) and IgG1 Fc fragment targeting VEGF-A, VEGF-B, and PGF, and currently approved by FDA to treat AMD and metastatic colorectal cancer [Holash et al. (2002) Proc. Natl. Acad. Sci. USA 99(17):11393-8].

Angiopoietins (ANGs) are a family of vascular growth factors also shown to play a critical role in angiogenesis [Alves et al. (2010) BMC Infect Dis 10:143-52]. Among which, ANG1 and ANG2 were demonstrated to be involved in balancing vessel stability and regression during both developmental and tumor-induced angiogenesis through the ANG-TIE signaling pathway [Augustin et al. (2009) Nat Rev Mol Cell Biol 10(3):165-77]. ANG1 and ANG2 bind to TIE2 with similar affinity [Suri et al. (1996) Cell 87(7):1171-80; Maisonpierre et al. (1997) Science 277:55-60]. The TIE2 extracellular domain (ECD) contains three EGF-like modules that are flanked by two immunoglobulin (Ig)-like domains and a third Ig-like domain followed by three fibronectin type III (FNIII) repeats. Structure and function analysis revealed the ANG-binding site was located to the N-terminal pair of Ig-like subdomains, however, it was shown to have less binding activity as compared with that of entire TIE2 ECD [Lin et al. (1997) J Clin Invest 100:2072-2078]. Recent studies indicated ANG1 is an agonist promoting stabilization of vessels by maximizing interactions between endothelial cells and their surrounding support cells and the extracellular matrix. ANG2, on the other hand, was found to be a context-dependent antagonist that promotes angiogenic sprouting or vessel regression [Maisonpierre et al. (1997) Science 277:55-60]. Circulating ANG2 levels have been identified as factor predicting poor prognosis in many human cancers, including metastatic melanoma, colorectal cancer (CRC), and chronic lymphocytic leukemia [Eklund et al. (2013) Exp Cell Res 319(9):1271-80]. The elevated ANG2 was reported to cause endothelial destabilization, leading to vessel regression, hypoxia, and increased expression of both ANG2 and VEGF, which together induced robust angiogenesis in the tumor [Holash et al. (1999) Science 284(5422): 1994-8].

However, it is desirable to develop a new approach for inhibition of angiogenesis in treatment of angiogenesis-associated diseases.

BRIEF SUMMARY OF THE INVENTION

Accordingly, the present invention provides a fusion protein that inhibits an ANG-TIE signaling pathway and a VEGF-VEGFR signaling pathway, wherein the fusion protein contains an ANG inhibiting domain and a VEGF inhibiting domain thereby providing a significantly improved efficacy in inhibition of angiogenesis through blocking the interactions between ANG, VEGF, or both with their receptors.

In one aspect, the invention provides a bi-functional fusion protein comprising one or more ANG binding motif containing fragments and one or more VEGF binding motif containing fragments, which are fused with a linker, thereby providing a significantly improved efficacy in inhibition of angiogenesis through blocking the interactions between ANG, VEGF, or both with their receptors.

In one embodiment of the invention, the bi-functional fusion protein comprises (1) one or more ANG binding motif containing fragments and (2) one or more VEGF binding motif containing fragments, which are fused with a linker.

In one example of the invention, the ANG binding motif containing fragment is a fragment antigen-binding (Fab) fragment, and the VEGF binding motif containing fragment is a single chain antibody (ScFv)

In one embodiment of the invention, the bi-functional fusion protein is (AT)a-Fc-(VT)b, simultaneously targeting vascular endothelial growth factor (VEGF) and angiopoietins (ANGs), which provides a significantly improved anti-angiogenesis efficacy, wherein AT is an ANG binding motif, VT is a VEGF binding motif; Fc is a linker; each of a and b is an integer from 1 to 10.

In one embodiment of the invention, ANG binding motif is a fragment consisting of Tie2 extracellular domain (ECD) amino acid residues from 23-448.

In one embodiment of the invention, the VEGF binding motif is VEGFR1 ECD D2, VEGFR2 ECD D3, or a chimeric domain containing the both motifs. In one preferred embodiment, the VEGF binding motif is a chimeric domain containing VEGFR1 ECD D2 and VEGFR2 ECD D3.

In one embodiment of the invention, the linker is IgG1 Fc fragment, a short flexible GS linker or combination thereof.

In one embodiment of the invention, the bi-functional protein is a fusion protein, (AT)-Fc-(VT), wherein AT is an AT fragment containing a Tie2 ECD amino acid residues from 23-448, Fc is an IgG Fc fragment, and VT is a VT fragment containing a chimeric domain containing VEGFR1 D2 and VEGFR2 D3.

In one example of the invention, the AT fragment and IgG Fc fragment are fused, and then linked with the VT at the C-terminal of the AT-Fc fragment by a short flexible GS linker.

In one specific embodiment, the bi-functional fusion protein is constructed by a fragment antigen-binding (Fab) containing the ANG inhibiting domain and a single chain antibody (ScFv) having a VEGF inhibiting domain. Preferably, the Fab is an anti-Ang2 Fab, and the ScFv is a fusion fragment consisting of Lucentis Vh and Vk.

In one embodiment, the Fc fragment is a wild-type or a variant of any human immunoglobulin isotype, subclass, or allotype.

In a further aspect, the present invention provides a pharmaceutical composition comprising the fusion protein disclosed herein and a pharmaceutically acceptable carrier.

According to the invention, the pharmaceutical composition is used for preventing or treating an angiogenesis-associated disease, such as age-related macular degeneration (AMD) disease or a primary or metastatic cancer.

In a further yet aspect, the present invention provides a fusion protein capable of binding to angiopoietins, which contains Tie2 ECD amino acid residues from 23 to 448 fused with IgG1 Fc fragment.

In a further yet aspect, the present invention provides a fusion protein against VEGF, which contains a chimeric motif containing VEGFR1 D2 and VEGFR2 D3 fused with an IgG1 Fc fragment at the C-terminal thereof.

It is to be understood that both foregoing general description and the following detail description are exemplary and explanatory and are not restrictive of the invention.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

The patent or application file contains at least one color drawing. Copies of this patent or patent application publication with color drawing will be provided by the USPTO upon request and payment of the necessary fee.

The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there are shown in the drawings embodiments which are presently preferred.

In the drawings:

FIG. 1A and FIG. 1B show the structure diagrams of AT-Fc and Fc-VT proteins, respectively, in which the AT-Fc protein comprises a synthetic nucleotide sequence having the Tie2 ECD amino acid residues from 23 to 448 that contains the three Ig-like domains and three EGF-like repeats fused to the N-terminal of IgG Fc to construct the expression vector for AT-Fc protein; and the VT-Fc protein comprises the chimeric VEGF binding motifs consisting of VEGFR1 ECD D2 and VEGFR2 ECD D3, which was synthesized and fused to the C-terminal of the IgG Fc with a GS linker in between to create the expression vector for Fc-VT protein.

FIG. 2A and FIG. 2B show the PAGE analyses of the AT-Fc protein and the Fc-VT protein, respectively, wherein the AT-Fc and Fc-VT expression vectors were used to transfect HEK293 cells, separately, which were expressed and then purified from the cell culture supernatant using Protein G chromatography. The purified proteins (3 μg/lane) were gel-analyzed under reducing or non-reducing condition. FIGS. 2A and 2B show the results of the AT-Fc and Fc-VT proteins that were properly dimerized with a molecular weight about 160 kD and 130 kD, respectively, and greater than 90% purity were obtained by the single step Protein G chromatography to purify both fusion proteins. Lane 1, non-reduced; Lane 2, reduced.

FIG. 3 shows the structure diagram of AT-Fc-VT and Tc350-253 bi-functional fusion proteins providing improved anti-angiogenesis activity.

FIG. 4 shows the purified AT-Fc-VT bi-functional fusion protein, wherein the AT-Fc-VT expression vector was used to transfect HEK293 cells. Expressed soluble AT-Fc-VT fusion proteins were purified from cell culture supernatant using Protein G chromatography. The purified proteins (3 μg/lane) were then reduced or non-reduced before gel analysis. Results indicated fusion proteins were properly dimerized with molecular weight about 250 kD and >90% purity can be obtained by single step Protein G chromatography. Lane 1, non-reduced; Lane 2, reduced.

FIG. 5 shows the purified Tc350-253 bi-functional fusion protein. Tc350-253 protein was transiently expressed by HEK293 cells and purified from the transfected cell culture supernatant via nickel affinity chromatography. The purified protein (5 μg/lane) was gel-analyzed under reducing or non-reducing condition. The Tc350-253 bi-functional protein was properly expressed with a molecular weight about 75 kD, and greater than 90% purity was obtained. NR, non-reduced; R, reduced.

FIG. 6 shows the binding activity of the AT-Fc protein against ANG1 and ANG2 in direct ELISA assays. The ligand pre-coated wells were first incubated with various concentrations of purified AT-Fc protein. The bound proteins were then detected with HRP conjugated goat anti-human IgG Fc specific antibody and OD₄₅₀ readings were plotted; and the results indicated AT-Fc binds to ANG1 or ANG2 with EC₅₀ of 4.26 nM and 2.01 nM, respectively.

FIG. 7 shows the binding activity of the Fc-VT protein against VEGF-A. VEGF-A pre-coated wells were first incubated with various concentrations of purified Fc-VT proteins. The bound proteins after washing were detected with HRP conjugated goat anti-human IgG Fc specific antibody and OD₄₅₀ readings were plotted, and the results indicated Fc-VT binds to VEGF-A with EC₅₀ of 0.33 nM.

FIGS. 8 and 9 show the direct ligand binding activity of purified AT-Fc-VT fusion protein against ANG2 and VEGF-A, respectively. Ligand pre-coated wells were first incubated with various concentrations of purified AT-Fc-VT protein. The bound proteins were then detected with HRP conjugated goat anti-human IgG Fc specific antibody and OD₄₅₀ readings were plotted. The results indicated AT-Fc-VT is able to bind ANG2 and VEGF-A with EC₅₀ of 2.57 nM and 0.35 nM, respectively, which was comparable to the binding activity of AT-Fc (0.28 nM) or Fc-VT (0.46 nM).

FIG. 10 shows the inhibitory effect of fusion proteins on VEGF-dependent HUVEC cell growth assay. Human umbilical vein endothelial cells are used to demonstrate VEGF-dependent cell growth inhibition by binding of fusion proteins, Fc-VT and AT-Fc-VT, to VEGF. In the experiment, HUVECs are maintained in the Endothelial Cell Growth Medium (Lonza, Inc.) with 2% FBS. A 96-well flat bottom microtiter plate is coated with collagen, and then incubated with 50 μl of 1 nM of VEGF-A (R&D systems, Inc.) and various concentrations of fusion proteins, Fc-VT or AT-Fc-VT, in each well for 1 hour at 37° C. before adding 50 μl of HUVECs at 1×10⁵ cells/ml in Medium-199 (10% FBS, Hyclone, Inc.). After incubate for 72 hours at 37C with 5% CO2, cell proliferation was assayed by adding 10 μl of MTS detection reagent (Promega Inc.) to each well and then measuring OD absorption at 450 /650 nm. In this experiment. Results indicated both Fc-VT and AT-Fc-VT have similar inhibitory activities against VEGF as comparable with Fc-VT (0.27 nM vs. 0.2 0 nM). In this study, a positive control, VEGFR1 ECD D2 and VEGFR2 ECD D3 fused C-terminally with IgG1 Fc fragment, was also included and shown to have IC₅₀ of 0.19 nM.

FIG. 11 shows the soluble ANG2 binding analysis. The purified fusion proteins, AT-Fc and AT-Fc-VT, were used to examine the ANG2 binding activity in this assay. The 100 μl of AT-Fc or AT-Fc-VT (1 μg/ml) fusion proteins was coated onto 96-well ELISA plates (NUNC®) for overnight at 4° C. The coated wells were blocked with 1% BSA in PBS for 1 hour at room temperature (RT). Three-fold serial diluted AT-Fc or AT-Fc-VT starting from 100 nM were incubated with equal volume of 500 pM recombinant ANG2 (PEPROTECH®) for 2hr at RT before transferred to 96-well with coated protein. After incubation, transferred sample to each respective well and incubated for 1 hr at RT. The plate was washed three times with washing buffer (ABCAM®) and incubated with Biotinylated anti-ANG2 antibody (ABCAM®) for 1 hr at RT. After washing, the plate was incubated with streptavidin-HRP (ABCAM®) for 45min at RT. After final washing, tetramethylbenzidine (TMB) was added and absorbance was measured at 450 nm using a plate reader. Data were analyzed using Prism (GRAPHPAD®) to calculate percentage of inhibition.

FIG. 12 shows the inhibition of VEGF-induced endothelial cell migration by bi-functional fusion protein.

FIG. 13 shows the inhibition of VEGF-induced endothelial cell tube formation by bi-functional fusion protein.

FIG. 14 shows the inhibition of ANG2-induced endothelial tube formation by bi-functional protein Tc350-253.

FIG. 15 shows the inhibition of VEGF-induced endothelial cell invasion by bi-functional angiogenesis blockers.

FIG. 16 shows the inhibition of VEGF/ANG2-induced endothelial cell tube formation by bi-functional protein T350-253.

FIG. 17 shows the inhibition of laser-induced choroidal neovascularization (CNV) in mice by bi-functional proteins.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise defined herein, scientific and technical terms used herein have the meanings that are commonly understood by those of ordinary skill in the art.

As used herein, the term “antibody” is a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes. A typical antibody is a tetramer that is composed of two identical pairs of polypeptide chains, each pair having one “light” and one “heavy” chain.

The term “antibody variable region” as used herein refers to an N-terminal region of an antibody variable chain comprising amino acid residues that are primarily responsible for antigen recognition. Those of ordinary skill in the art are readily able to identify an antibody variable region and to determine the minimum size needed to confer antigen recognition. The antibody variable region may be a single chain antibody, for example a single chain Fv antibody (scFv) in which a variable heavy chain region and a variable light chain region are joined together (directly or through a peptide linker) to form a continuous polypeptide. The scFv antibody may be chemically synthesized or may be expressed from a nucleic acid including V_(H)- and V_(L)-encoding sequences either joined directly or joined by a peptide-encoding linker.

The invention provides a bi-functional fusion protein that inhibits an angiopoietin (ANG)-TIE signaling pathway and a vascular endothelial growth factor (VEGF)-vascular endothelial growth factor receptor (VEGFR) signaling pathway. In the invention, the fusion protein contains an ANG inhibiting domain and a VEGF inhibiting domain thereby providing a significantly improved efficacy in inhibition of angiogenesis through blocking the interactions between ANG, VEGF, or both with their receptors.

In the invention, the both VEGF and ANG playing a crucial role in the process of angiogenesis, were used for construction of IgG Fc fusion blockers. The AT-Fc and Fc-VT proteins having ANG and VEGF binding activity, respectively, were first generated, and AT-Fc-VT bi-functional protein was created to provide a bi-functional fusion protein (with ANG and VEGF binding activities) accordingly. These AT-Fc and Fc-VT proteins were shown to be able to bind its respective ligands with high affinity, and also to provide anti-ANG and anti-VEGF functions in cell-based assays, respectively. In the invention, the Fc fusion protein contains the domains of human proteins and are of all human origins, and are expected to be non-immunogenic, and thus, potentially can be further developed as therapeutics for treating angiogenesis-associated diseases.

In the invention, any motif, peptide, protein, or fragment having blocking VEGF activity may be used, for example anti-VEGF antibodies, VEGF traps, or VEGF receptor (VEGFR) extracellular Ig domains such as D1-D7, in particular, VEGFR1 extracellular Ig domain 2 (ECD, D2), VEGFR2 extracellular Ig domain 3 (ECD, D3).

In the invention, any motif, peptide, protein, or fragment binding to angiopoietin (ANG) may be used, for example, ANG1 and ANG2 binding to TIE2. The TIE2 ECD contains three EGF-like modules that are flanked by two immunoglobulin (Ig)-like domains and a third Ig-like domain followed by three fibronectin type III (FNIII) repeats. The ANG-binding site was located to the N-terminal pair of Ig-like subdomains.

Since VEGF and ANG2 were upregulated in a variety of cancer cell types, blocking the ANG-TIE interaction together with/without VEGF blocker, a bi-functional fusion against ANG2 and VEGF is created in this invention for treatment of cancers and age-related macular degeneration (AMD) disease.

In various embodiments, angiogenesis-associated diseases may be age-related macular degeneration (AMD) disease, or a primary and metastatic cancer.

The age-related macular degeneration (AMD) disease is a disease that blurs the sharp, central vision as needed for “straight-ahead” activities such as reading, sewing and driving. Normally, AMD affects the macula, the part of an eye.

The primary and metastatic cancers may be carcinomas of breast, colon, rectum, lung, oropharynx, hypopharynx, esophagus, stomach, pancreas, liver, gallbladder and bile ducts, small intestine, urinary tract (including kidney, bladder and urothelium), female genital tract (including cervix, uterus, and ovaries as well as choriocarcinoma and gestational trophoblastic disease), male genital tract (including prostate, seminal vesicles, testes and germ cell tumors), endocrine glands (including the thyroid, adrenal, and pituitary glands), and skin, as well as hemangiomas, melanomas, sarcomas (including those arising from bone and soft tissues as well as Kaposi's sarcoma), tumors of the brain, nerves, eyes, and meninges (including astrocytomas, gliomas, glioblastomas, retinoblastomas, neuromas, neuroblastomas, Schwannomas, and meningiomas), tumors arising from hematopoietic malignancies, acute leukemia, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), B cell lymphoma, Burkitt's lymphoma, chronic myelocytic leukemia (CIVIL), chronic lymphocytic leukemia (CLL), hairy cell leukemia, Hodgkin's and non-Hodgkin's lymphomas, hematopoietic malignancies, Kaposi's sarcoma, malignant lymphoma, malignant histiocytosis, malignant melanoma, multiple myeloma, paraneoplastic syndrome/hypercalcemia of malignancy, or solid tumors.

In one embodiment of the invention, a fragment having Tie2 ECD amino acid residues from 23 to 448 (SEQ ID NO: 1) was used to generate an AT-Fc protein with IgG1 Fc fragment (SEQ ID NO: 2) at C-terminal to engineer an AT-Fc protein.

In another embodiment of the invention, a fragment containing the VEGFR1 D2 and VEGFR2 D3 chimeric motifs (SEQ ID NO: 3) was fused at the C-terminal of the IgG1 Fc fragment (SEQ ID NO: 2) to construct an Fc-VT protein with a short flexible GS linker (SEQ ID NO: 4) between them to ensure that the folding is correct and steric hindrance is minimized.

In one yet embodiment, the AT-Fc-VT fusion protein was engineered through fusion to obtain a domain containing VEGFR1 ECD D2 and VEGFR2 ECD D3 (SEQ ID NO: 3) at C-terminal of AT-Fc also included with a GS linker (SEQ ID NO: 4) between the AT-FC and VT proteins.

In a specific embodiment, the bi-functional protein Tc350-253 was engineered and expressed as a disulfide-linked Tc350-Tc253 protein, wherein Tc350 was constructed by fusing Lucentis variable heavy chain domain (Vh) (SEQ ID NO: 5) and variable kappa domain (Vk) (SEQ ID NO: 6) at the C-terminal of Anti-Ang2-Fragment antigen-binding (Fab) heavy chain (HC) (SEQ ID NO: 7) with the short flexible GS linker (SEQ ID NO: 4) in between, and Tc253 was constructed to have Anti-Ang2-Fab light chain (LC) fragment (SEQ ID NO: 8).

In further embodiment, all fusion proteins described in this invention were leaded by a signal peptide (SEQ ID NO: 9) for the secretion of expressed proteins.

The amino acid sequences of the AT-Fc, Fc-VT, AT-Fc-VT, and Tc350 were those set forth in SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13, respectively.

In the present invention, the above-mentioned AT-Fc and/or Fc-VT proteins were transiently expressed by HEK293 cells and purified from the transfected cell culture supernatant via Protein G chromatography. It was found that the products having a purity greater than 90% were obtained in a single step purification process and all fusion proteins were properly formed and expressed.

According to the present invention, the bi-functional fusion protein, AT-Fc-VT, simultaneously targeting VEGF and ANGs was created through fusing the VEGF binding motif at the C-terminal of the ANG protein. As shown in the results, the AT-Fc-VT fusion protein according to the invention provided a bi-functional efficacy in inhibiting VEGF-dependent HUVEC cell proliferation, cell migration, tube formation as well as to bind ANG2 protein, as comparable with Fc-VT or AT-Fc protein respectively.

According to the present invention, the bi-functional protein Tc350-253 provides a beneficial effect on VEGF and/or ANG2-induced angiogenesis-associated phenotypes. Particularly, the bi-functional protein Tc350-253 unexpectedly improves laser-induced choroidal neovascularization, which has been used extensively in studies of the exudative form of age-related macular degeneration (AMD).

So that the disclosure may be more readily understood, select terms are defined below.

As used herein, the term “fusion protein” refers to a protein created through the connection of two or more binding proteins, or motifs, or peptides/amino acid fragments coding for different genes, the translation of which genes results in a single or multiple polypeptides with multi-functional properties derived from each of the original proteins. A fusion protein can include a protein conjugated to an antibody, an antibody conjugated to a different antibody, or an antibody conjugated to a Fab fragment.

Each of the AT or VT fragment is an antibody, or a fragment of antibody with long half-life in vivo.

As used herein, the term “linker” refers to an amino acid residue or fragment, or a polypeptide comprising two or more amino acid residues joined by peptide bonds that are used to link two peptides, polypeptides or proteins. The linker may be a Fc fragment, which is an immunoglobulin Fc region of a wild-type or a variant of a human immunoglobulin isotype, subclass, or allotype thereof.

The term “Fc region” defines the C-terminal region of an immunoglobulin heavy chain of an antibody. The Fc region may be a native sequence Fc region or a variant Fc region.

As used herein, the term “pharmaceutical composition” refers to a composition or a formulation comprising the fusion protein according to the invention, which can be prepared by mixing the fusion protein according to the invention with optional pharmaceutically acceptable carriers, including solvents, dispersion media, isotonic agents and the like. The carrier can be liquid, semi-solid or solid carriers. In some embodiments, carriers may be water, saline solutions or other buffers (such as serum albumin and gelatin), carbohydrates (such as monosaccharides, disaccharides, and other carbohydrates including glucose, sucrose, trehalose, mannose, mannitol, sorbitol, or dextrins), gel, lipids, liposomes, resins, porous matrices, binders, fillers, coatings, stabilizers, preservatives, antioxidants (including ascorbic acid and methionine), chelating agents (such as EDTA), salt forming counter-ions (such as sodium), non-ionic surfactants [such as TWEEN®, PLURONICS® or polyethylene glycol (PEG)], or combinations thereof.

In some embodiments, the pharmaceutical composition further comprises one additional therapeutic agent for preventing or treating said disease or disorder to be treated. For example, the additional agent may be a therapeutic agent, a chemotherapeutic agent; an imaging agent, a cytotoxic agent, an angiogenesis inhibitor, a kinase inhibitor, a co-stimulation molecule blocker (including but not limited to anti-B7.1, anti-B7.2, CTLA4-Ig, anti-CD20), an adhesion molecule blocker (including but not limited to an anti-LFA-1 antibody, an anti-E/L selectin antibody, a small molecule inhibitor), and an anti-cytokine antibody or functional fragment thereof (including but not limited to an anti-IL-18, an anti-TNF, and an anti-IL-6/cytokine receptor antibody).

The invention is further illustrated by the following examples, which should not be construed as further limiting.

EXAMPLES Example 1 Expression and Purification of Angiogenesis Blockers

The TIE2 ECD amino acid residues from 23-448 was used as ANG binding motif, and the chimeric domains with VEGFR1 D2 and VEGFR2 D3 were used as VEGF binding trap. The corresponding DNAs were synthesized and fused with IgG1 Fc fragment gene at C-terminal for ANG blocker (AT-Fc) (FIG. 1A) or at N-terminal for VEGF blocker (Fc-VT) (FIG. 1B) with a short flexible GS linker was placed in between. To construct bi-functional AT-Fc-VT expression vector, AT-Fc was fused with GS linker and the VEGF motif, sequentially, at C-terminal (FIG. 3 ). These fusion constructs were cloned to pCI-neo (Promega, Inc.) expression vector between the EcoR I and Not I restriction sites under the control of a CMV promoter. The vector contains a Neomycin resistant gene and an Ampicillin resistant gene for mammalian cell selection and E. coli propagation. All fusion constructs contained a signal peptide at the N-terminal for the secretion of soluble fusion proteins. These expression constructs were used to transfect 50 ml of HEK293 cells at density of 1×10⁶ cells per ml transiently. The culture media was harvested after 96 hours of transfection and fusion proteins were purified via Protein G chromatography.

As shown in FIGS. 2 and 4 , about 3 mg fusion proteins (per transfection) having a purity greater than 90% was obtained.

To construct bi-functional protein Tc350-253 expression vector (FIG. 3 ), Anti-Ang2 Fab HC was sequentially fused with Lucentis Vh and Lucentis Vk separated by GS linker. Tc350-253 protein was transiently expressed by HEK293 cells and purified from the transfected cell culture supernatant via nickel affinity chromatography. The purified protein (5 μg/lane) was gel-analyzed under reducing or non-reducing condition. The Tc350-253 bi-functional protein was properly expressed with a molecular weight about 75 kD, and greater than 90% purity was obtained (FIG. 5 ).

Example 2 In Vitro Binding of Angiogenesis Blockers

Overnight ligand pre-coated (100 ng/well) wells were washed 3 times with 1× PBS and blocked with 400 u1 5% non-fat skin milk in PBS for 2 hr. After blocking, 100 ul of 3-fold serially diluted purified protein started from 30 nM were added to each corresponding well in duplicate and incubated for 1hr at room temperature on shaker. After binding, wells were washed 3 times with PBST (1× PBS with 0.1% TWEEN-20®) before adding 100 ul of 1:2500 diluted goat anti-human IgG Fc HRP conjugates (Jackson ImmunoResearch Inc.) to each well and incubated on shaker for another hour. After final washing, TMB reagent (Invitrogen, Inc.) was added and OD absorption at 450 nm was measured. The data analyzed by sigmoidal curve fitting using Prism 4. Binding of AT-Fc, Fc-VT, and AT-Fc-VT to ANG or VEGF were assessed by similar assays.

As shown in FIG. 6 , the calculated EC₅₀ for AT-Fc against ANG1 and ANG2 are 4.26 nM and 2.01 nM, respectively. The results collected herein indicated the soluble extracellular domain (amino acid residues 23-448) is functionally active in angiopoietin binding and could modulate the activity of the membrane-bound form by competing for angiopoietins.

As shown in FIG. 7 , the Fc-VT had the anti-VEGF efficacy in the VEGF-A binding assessment. The calculated EC50 against VEGF-A was 0.33 nM. As shown in the results as collected, the C-terminally localized VEGFR1 D2 and VEGFR2 D3 chimeric VEGF trap was functional with high affinity and could neutralize VEGF's biological activities.

As shown ion FIGS. 8 and 9 , the binding activities to ANG2 and VEGF of the purified AT-Fc-VT bi-functional fusion protein were assessed and the results indicated that the AT-Fc-VT fusion protein was able to bind ANG2 and VEGF-A with EC₅₀ of 2.57 nM and 0.35 nM, respectively, and provided significantly improved binding activities as compared with the AT-Fc against ANG2, and the Fc-VT against VEGF.

In conclusion, the AT-Fc-VT fusion protein were confirmed to have bi-functions to modulate the ANG-TIE and VEGF-VEGFR pathways through the inhibition of ligand binding and receptor activation.

Example 3 Inhibition of VEGF-Dependent HUVEC Proliferation Assay by Angiogenesis Blockers

The Fc-VT and AT-Fc-VT were used to inhibit VEGF activities in a cell base assay. Human Umbilical Vein Endothelial Cells (HUVEC cells, Lonza, Inc.) were used to demonstrate VEGF-dependent cell proliferation which can be inhibited by VEGF blocker. In the experiment, HUVECs were maintained in the Endothelial Cell Growth Medium (Lonza, Inc.) with 2% FBS. Collagen pre-coated wells were loaded with 50 ml of 1 nM of VEGF-A (R&D systems, Inc.) and various concentrations of Fc-VT or AT-Fc-VT per well for 1 hour at 37° C. before adding 50 ml of HUVECs at 1×10⁵ cells/ml in Medium-199 (10% FBS, Hyclone, Inc.) to each well. After 72 hours incubation at 37C with 5% CO2, the cell growth was assayed by adding 10 ml MTS detection reagent (Promega Inc.) to each well and then measuring OD absorption at 450/650 nm.

As shown in FIG. 10 , the 50 percent inhibitory activity (IC₅₀) of AT-Fc-VT on VEGF-dependent HUVEC cell proliferation assay was 0.26 nM, similar to that of Fc-VT (0.20 nM) in this example.

Example 4 Inhibition of Endothelial Cell Tube Formation by Angiogenesis Blockers

The AT-Fc and AT-Fc-VT were used to determine the anti-ANG2 activities in the tube formation assay. Human umbilical vein endothelial cells were used to demonstrate Ang2-dependent cell tube formation which can be inhibited by binding of AT-Fc or AT-Fc-VT to Ang2. HUVECs are maintained in the Endothelial Cell Growth Medium (Lonza, Inc.) with 2% FBS. Prior to assay, HUVEC cells were cultivated in basal media without serum for 2 hr, and then trypsinized and re-suspended in 1% serum at 10⁵cells/ml. 1.5×10⁴ cells were seeded per Matrigel pre-coated well, and 200 ng/ml Ang2 with/without AT-Fc or AT-Fc-VT at various concentrations. After incubation at 37° C. for 18 hr, document tube formation with 3 non-overlapping images taken from each well repeated in triplicates (9 images/sample). The tube length was calculated using the ImagePro software (Media Cybernetics Inc.).

As shown in FIG. 11 , the 50 percent inhibitory activity (IC₅₀) of AT-Fc-VT on tube formation was 2.45 nM, less than that of AT-Fc (3.68 nM) in this example.

Example 5 Bi-Functional Fusion Protein AT Significantly Inhibits VEGF-Induced Endothelial Cell Migration

The inhibitory effect of the bi-functional protein AT-Fc-VT on VEGF-induced chemotaxis was assessed using HUVECs on transwell plates (8-μm pore size, Costar). HUVEC cells (1×10⁵) with M199 serum-free medium were placed in the upper well. The ligand human VEGF (0.2 μg/ml) were mixed with Eylea (2 μg/ml) or AT-Fc-VT (2 μg/ml) in 10% FBS M199 medium and placed in the lower well. The transwell plate were incubated for 24 h in a 5% incubator to allow cells from the upper well to transmigrade towards the bottom chamber. The membrane inserts were then fixed and stained with 1% crystal violet. Cells adhered to the lower surface of membrane inserts were visualized via microscopy and the average number of migrated cells was calculated from five representative fields on triplicate inserts using ImageJ software.

We examined the effect of AT-Fc-VT protein on the physiological responses of endothelial cells to VEGF stimulation. HUVEC cells were added to the upper chambers and were exposed to VEGF with vehicle, VEGF with Eylea, VEGF with AT-Fc-VT, individually. After 24 h, cells that migrated to the lower chamber of the membrane were counted. VEGF significantly increased the migration of HUVEC cells. However, the angiogenesis blocker AT-Fc-VT significantly inhibited the migration induced by VEGF (FIG. 12 ).

Example 6 Bi-Functional Fusion Protein AT Inhibits VEGF-Induced HUVEC Cells Tube Formation

To study the function of hi-functional angiogenesis blocker AT-Fc-VT in angiogenesis, an in vitro Matrigel tube formation assay was performed in human umbilical vein endothelial cells. HUVEC cells were incubated in basal media without serum for 2 hr, and then trypsinized by accutase. 4×10³ HUVEC cells were seeded onto Matrigel pre-coated wells containing VEGF (1 μg/ml) with AT-Fc-VT (100 μg/ml) or Eylea (100 μg/ml) at 37° C. for 4.5 hr. Quantification of endothelial network formation was performed by Image J angiogenesis system (5 images/sample).

Under VEGF treatment, when cultured on Matrigel for 4.5 hr, HUVEC cells displayed a primary a vascular tubular network. However, bi-functional protein AT-Fc-VT significantly disrupted the formation of tubular structures (FIG. 13 ).

Example 7 Bi-Functional Protein Tc350-253 Inhibits ANG2-Induced Tube Formation of HUVEC Cells

HUVEC cells were incubated in basal media without serum for 2 hr. 4×10³ HUVEC cells were seeded onto Matrigel pre-coated wells containing ANG2 (0.5 μg/ml) with Tc350-253 (50 μg/ml) or Tc293-240 (50 μg/ml) at 37° C. for 4.5 hr. Quantification of endothelial network formation was performed by Image J angiogenesis system (5 images/sample).

Higher levels of ANG2 correlate with disease severity in human wet Age-related Macular Degeneration (AMD) and cancer progression including angiogenesis, metastasis and inflammation. We generated an innovative design to co-targeting VEGF and Ang2. To address the function of bi-functional protein in angiogenesis, an in vitro Matrigel tube formation assay was performed. The result indicated that bi-functional proteins Tc350-253 or Tc293-240 suppress ANG2-induced HUVEC cells tube formation in vitro (FIG. 14 ).

Example 8 Bi-Functional Proteins AT and Tc350-253 Inhibit VEGF-Induced HUVEC Cell Invasion

The invasion assay was performed by precoating the transwell inserts with Matrigel Basement Membrane Matrix (BD Biosciences, San Diego, Calif., USA), according to the manufacturer's instructions. HUVEC cells (2×10⁵) with Medium-199 were placed in the upper well. The ligand human VEGF (0.2 μg/ml) were mixed with Eylea (2 μg/ml), AT-Fc-VT (2 μg/ml) or Tc350-253 (2 μg/ml) in the lower chamber, individually. The transwell plate were incubated for 24 h in a 5% incubator to allow cells from the upper well to transmigrate towards the bottom chamber. The membrane inserts were then fixed and stained with 1% crystal violet. Cells adhered to the lower surface of membrane inserts were visualized via microscopy and the average number of migrated cells was calculated using ImageJ software.

The bi-functional proteins of AT-Fc-VT and Tc350-253 were used to determine the anti-VEGF activities using boyden chamber invasion assay. Similar to the results of the migration study, the angiogenesis blockers AT-Fc-VT and Tc350-253 inhibited the invasive ability induced by VEGF (FIG. 15 ).

Example 9 Bi-Functional Protein Tc350-253 Inhibits VEGF/ANG2-Induced HUVEC Cells Tube Formation

HUVEC cells were incubated in basal media without serum for 2 hr, and then trypsinized by accutase. 4×10³ HUVEC cells were seeded onto Matrigel pre-coated wells containing VEGF (0.5 μg/ml)/ANG2 (0.5 μg/ml) with Eylea (50 μg/ml), Tc350-253 (50 μg/ml) or other anti-Ang2 antibodies (50 μg/ml) individually at 37° C. for 4.5 hr. Quantification of endothelial network formation was performed by Image J angiogenesis system (5 images/sample).

To study the function of dual-targeting protein Tc350-253 in angiogenesis, an in vitro Matrigel tube formation assay was performed in human umbilical vein endothelial cells. Bi-functional angiogenesis blocker Tc350-253 significantly disrupted the formation of tubular structures (FIG. 16 ). The bioactivity of bi-functional protein Tc350-253 was better than Eylea or anti-Ang2 antibodies (Nesvacumab and Tc252-253) in this assay.

Example 10 Bi-Functional Proteins AT and Tc350-253 Inhibit Neovascularization in Laser-Induced Choroidal Neovascularization (CNV) Mouse Model

Laser-induced CNV was used animal model for wet AMD to assess the activities of angiogenesis blockers. After mice were anesthetized with ketamine hydrochloride (100 mg/kg body weight) and the pupils were dilated with 1% tropicamide, three burns of 532 nm diode laser photocoagulation were delivered to each retina. Burns were performed in the 9, 12, and 3 o'clock positions of the posterior pole of the retina. Production of a bubble at the time of laser, indicating rupture of Bruch's membrane, was an important factor in obtaining CNV.

In the laser-induced CNV model, male C57BL/6 mice were intravitreally injected with vehicle control, EYLEA® (40 μg), AT-Fc-VT (40 μg) or Tc350-253 (40 μg) in the right eye on day 1 (n=5/group). The retinal angiogenesis was examined by fluorescein angiography and optical coherence tomography on day 0, 7, 14 and 21. For fluorescein angiography, 10% sodium fluorescein was injected intravenously and serial images were captured immediately (every 10 s for 10 min). The injected sodium fluorescein is completely excreted after 24 h. Images were captured using the Micron III retinal imaging microscope (Phoenix Research Laboratories, San Ramon, Calif., USA) to determine the extent of CNV formation. For OCT analysis, ellipsoid volume was calculated by the formula V=4/3πabc where a (width), b (depth) and c (length) are the radii of the horizontal plane or vertical plane of the ellipsoid.

VEGF and ANG-2 appear strongly linked to pathological neovascularization and vascular permeability, which are the hallmarks of ocular neovascular disease. We tested the effect of bi-functional proteins AT-Fc-VT or Tc350-253 on angiogenesis by laser-induced choroidal neovascularization mouse model. It showed the bi-functional proteins AT-Fc-VT and Tc350-253 significantly inhibit neovascularization in this model using fundus fluorescein angiography (FFA) analysis (FIG. 17 ). Quantification of laser-CNV lesions as ellipsoids by optical coherence tomography (OCT) also showed a significant reduction in lesion volume after bi-functional proteins AT-Fc-VT or Tc350-253 treatment (FIG. 17 ).

While the present invention has been disclosed by way preferred embodiments, it is not intended to limit the present invention. Any person of ordinary skill in the art may, without departing from the spirit and scope of the present invention, shall be allowed to perform modification and embellishment. Therefore, the scope of protection of the present invention shall be governed by which defined by the claims attached subsequently. 

What is claimed is:
 1. A bi-functional fusion protein that inhibits an angiopoietin (ANG)-TIE signaling pathway and a vascular endothelial growth factor (VEGF)-vascular endothelial growth factor receptor (VEGFR) signaling pathway by blocking the interaction of ANG with its receptor and/or VEGF with its receptor, wherein the fusion protein has the amino acid sequence set forth in SEQ ID NO:
 12. 2. A pharmaceutical composition comprising the bi-functional fusion protein of claim 1 and a pharmaceutically acceptable carrier.
 3. The pharmaceutical composition of claim 2, which is for preventing or treating age-related macular degeneration (AMD) disease.
 4. The pharmaceutical composition of claim 2, which is for inhibiting cancer angiogenesis. 